This invention relates to the use of compounds for the manufacture of a medicament for treating humans suffering from diabetes and insulin resistance. In particular, the invention relates to the pulmonary delivery of glucagon-like peptide-1 (GLP-1) and analogues thereof for systemic absorption through the lungs to eliminate the need for administering anti-diabetic compounds by injection.

Glucagon-like peptide-1 was first identified in 1987 as a incretin hormone, a peptide secreted by the gut upon ingestion of food. Glucagon-like peptide-1 is secreted by the L-cells of the intestine after being proteolytically processed from the 160 amino acid precursor protein, preproglucagon. Cleavage of preproglucagon first yields glucagon-like peptide-1, a 37 amino acid peptide that is poorly active. A subsequent cleavage of the peptide bond between residues 6 and 7 yields biologically active glucagon-like peptide-1 referred to as GLP-1(7-37). It should be noted that this specification uses the nomenclature scheme that has developed around this hormone. By convention in the art, the amino terminus of GLP-1(7-37) has been assigned number 7 and the carboxy terminus number 37. Approximately 80% of the GLP-1(7-37) that is synthesized is amidated at the C-terminal after removal of the terminal glycine residue in the L-cells. The biological effects and metabolic turnover of the free acid GLP-1(7-37), and the amide, GLP-1(7-36)NH₂, are indistinguishable. As used herein, these two naturally-occurring forms will be referred to collectively as GLP-1.

GLP-1 is known to stimulate insulin secretion (insulinotropic action) causing glucose uptake by cells which decreases serum glucose levels (see, e g., Mojsov, S., Int. J. Peptide Protein Research, 40:333-343 (1992)). Numerous GLP-1 analogues and derivatives demonstrating insulinotropic action are known in the art. Also it has been demonstrated that the N-terminal histidine residue (His 7) is very important to insulinotropic activity of GLP-1 (Suzuki, S., et al. Diabetes Res.; Clinical Practice 5 (Supp. 1):S30 (1988).

Multiple authors have demonstrated the nexus between laboratory experimentation and mammalian, particularly human, insulinotropic responses to exogenous administration of GLP-1. See, e.g., Nauck, M.A., et al., Diabetologia, 36:741-744 (1993); Gutniak, M., et al., New England J. of Medicine, 326(20):1316-1322 (1992); Nauck, M.A., et al., J. Clin. Invest., 91:301-307 (1993); and Thorens, B., et al., Diabetes, 42:1219-1225 (1993)].

GLP-1 based peptides hold great promise as alternatives to insulin therapy for patients with diabetes who have failed on sulfonylureas. GLP-1 has been studied intensively by academic investigators, and this research has established the following for patients with type II diabetes who have failed on sulfonylureas:

1. 1) GLP-1 stimulates insulin secretion, but only during periods of hyperglycemia. The safety of GLP-1 compared to insulin is enhanced by this property of GLP-1 and by the observation that the amount of insulin secreted is proportional to the magnitude of the hyperglycemia. In addition, GLP-1 therapy will result in pancreatic release of insulin and first-pass insulin action at the liver. This results in lower circulating levels of insulin in the periphery compared to subcutaneous insulin injections.
2. 2) GLP-1 suppresses glucagon secretion, and this, in addition to the delivery of insulin via the portal vein helps suppress the excessive hepatic glucose output in diabetic patients.
3. 3) GLP-1 slows gastric emptying which is desirable in that it spreads nutrient absorption over a longer time period, decreasing the postprandial glucose peak.
4. 4) Several reports have suggested that GLP-1 may enhance insulin sensitivity in peripheral tissues such as muscle and fat.
5. 5) Finally, GLP-1 has been shown to be a potential regulator of appetite.

- Meal-time use of GLP-1 based peptides offers several advantages over insulin therapy. Insulin therapy requires blood glucose monitoring, which is both expensive and painful. The glucose-dependency of GLP-1 provides an enhanced therapeutic window in comparison to insulin, and should minimize the need to monitor blood glucose. Weight gain also can be a problem with intensive insulin therapy, particularly in the obese type II diabetic patients.

The therapeutic potential for native GLP-1 is further increased if one considers its use in patients with type I diabetes. A number of studies have demonstrated the effectiveness of native GLP-1 in the treatment of insulin dependent diabetes mellitus. Similar to patients with type II diabetes, GLP-1 is effective in reducing fasting hyperglycemia through its glucagonostatic properties. Additional studies have indicated that GLP-1 also reduces postprandial glycemic excursions in type I patients, most likely through a delay in gastric emptying. These observations indicate that GLP-1 will be useful as a treatment in patients with type I and type II diabetes and in patients with impaired glucose tolerance.

To date administration of clinically proven peptide hormones and as well as GLP-1 has generally been accomplished by subcutaneous injection which is both inconvenient and unattractive. Therefore, many investigators have studied alternate routes for administering peptide hormones such as oral, rectal, transdermal, and nasal routes. Thus far, however, these routes of administration have not resulted in clinically proven peptide hormone therapy.

It has been known for a number of years that some proteins can be absorbed from the lung. For example, insulin administered by inhalation aerosol to the lung was first reported by Gaensslen in 1925. Despite the fact that a number of human and animal studies have shown that some insulin formulations can be absorbed through the lungs, pulmonary delivery of peptide hormones has not been vigorously pursued because of very low bioavailability. Larger proteins, such as cytokines and growth factors which are generally larger than 150 amino acid residues, are often readily absorbed by the cells lining the alveolar regions of the lung. Pulmonary absorption of smaller proteins is however much less predictable; though insulin (51 residues), calcitonin (32 residues) and parathyroid hormone (34 residues) have been reported to be systemically absorbed through the pulmonary route. See US Patent No: 5,607,915. Despite systemic absorption by the lung of some small protein hormones, the pharmacodynamics associated with pulmonary delivery of peptides is unpredictable.

Thus, there is a need to provide a reliable pulmonary method of delivering GLP-1 and related analogues because it would offer patients an attractive, non-invasive alternative to insulin. This need is particularly true since insulin has a very narrow therapeutic index while GLP-1 treatment offers a way to normalize blood glucose only in response to hyperglycemic conditions without the threat of hypoglycemia.

Not all protein hormones can be efficiently absorbed through the lungs, and there are many factors that affect it. Absorption of proteins in the lung is largely dependent on the physical characteristics of the protein. Thus, even though pulmonary delivery of some protein hormones has been observed, the physical properties and short length of GLP-1 and some related peptides made it unclear whether such peptides could be effectively delivered through the pulmonary route.

Efficient pulmonary delivery is dependent on the ability to deliver the protein to the deep lung alveolar epithelium. Protein particles that lodge in the upper airway epithelium are not absorbed to a significant extent because the overlying mucus functions to trap, and then clear debris by mucociliary transport up the airway. This mechanism is also a major contributor to low bioavailability. The extent to which proteins are not absorbed and instead eliminated by these routes depends on their solubility, their size, and other largely uncharacterized mechanisms.

Even when a peptide hormone can be reproducibly delivered to the deep lung alveolar epithelium, it is difficult to predict whether it will be rapidly absorbed and transported to the blood. Absorption values for some proteins delivered through the lungs have been calculated and range from fifteen minutes for parathyroid hormone (1-34) to 48 hours for glycosylated α1-antitrypsin. Moreover a variety of endogenous peptidases exist in the lung which can degrade peptides prior to absorption. Thus, the longer it takes for a peptide particle to dissolve and be absorbed, the greater the chance for enzymatic inactivation. Thus, because of the small size of GLP-1 and its inherent susceptibility to certain enzymes, it was most surprising to find that an aerosolized GLP-1 analogue could be reproducibly and effectively delivered through the lungs.

WO 93/18785 discloses medicaments for intranasal administration containing GLP-1(7-37), GLP-1(7-36)amide or derivatives or analogues thereof and a phospholipid. EP-A-0619322 discloses methods and compositions for the treatment of non-insulin dependent diabetes mellitus by prolonged administration of GLP-1(7-37) and related peptides. WO 98/08871 discloses derivatives of GLP-1 and analogues thereof having a lipophilic substituent. WO 98/19698 discloses the use of GLP-1 or analogues or derivatives thereof in methods and compositions for reducing body weight. WO 99/03454 (not relevant for inventive step) discloses a method for delivering a biologically active substance by the use of a biodegradable macromer.

The present invention relates to the use of a dipeptidyl peptidase IV protected glucagon-like peptide-1 (GLP-1) molecule for the manufacture of a medicament for the treatment of type II diabetes, hyperglycemia, or impaired glucose tolerance by pulmonary administration. Preferably, the glucagon-like peptide-1 molecule is delivered by inhalation and to the lower airway of the patient.

The glucagon-like peptide-1 can be delivered in a carrier, as a solution or suspension, or as a dry powder, using any of a variety of devices suitable for administration by inhalation. Preferably, the glucagon-like peptide-1 is delivered in a particle size effective for reaching the lower airways of the lung.

The term "GLP-1" refers to human glucagon-like peptide-1 whose sequences and structures are known in the art. See US Patent No. 5,120,712. As previously discussed, there are two native forms of human GLP-1, GLP-1(7-37) and GLP-1(7-36)NH₂ which will be distinguished only when necessary.

The term "GLP-1 analogue" is defined as a molecule having one or more amino acid substitutions, deletions, inversions, or additions compared with GLP-1. Many GLP-1 analogues are known in the art and include, for example, GLP-1(7-34) and GLP-1(7-35), GLP-1(7-36), Val⁸-GLP-1(7-37), Gln⁹-GLP-1(7-37), D-Gln⁹-GLP-1(7-37), Thr¹⁶-Lys¹⁸-GLP-1(7-37), and Lys¹⁸-GLP-1(7-37). GLP-1(7-34) and GLP-1(7-35) are disclosed in U.S. Patent No: 5,118,666.

The term "GLP-1 derivative" is defined as a molecule having the amino acid sequence of GLP-1 or a GLP-1 analogue, but additionally having chemical modification of one or more of its amino acid side groups, a-carbon atoms, terminal amino group, or terminal carboxylic acid group. A chemical modification includes, but is not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties. Modifications at amino acid side groups include, without limitation, acylation of lysine e-amino groups, N-alkylation of arginine, histidine, or lysine, alkylation of glutamic or aspartic carboxylic acid groups, and deamidation of glutamine or asparagine. Modifications of the terminal amino include, without limitation, the desamino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the terminal carboxy group include, without limitation, the amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications. Lower alkyl is C₁-C₄ alkyl. Furthermore, one or more side groups, or terminal groups, may be protected by protective groups known to the ordinarily-skilled protein chemist. The a-carbon of an amino acid may be mono- or di-methylated.

The term "GLP-1 molecule" means GLP-1, GLP-1 analogue, or GLP-1 derivative as defined in claim 1.

A preferred group of GLP-1 analogues is defined by the formula: and the pharmaceutically-acceptable salts thereof, wherein: R₁ is selected from the group consisting of L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, b-hydroxy-histidine, homohistidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine; X is selected from the group consisting of Gly, Val, Thr, Ile, and alpha-methyl-Ala; Y is selected from the group consisting of Glu, Gln, Ala, Thr, Ser, and Gly; Z is selected from the group consisting of Glu, Gln, Ala, Thr, Ser, and Gly; and R₂ is selected from the group consisting of NH₂, and Gly-OH.

Another preferred group of compounds consistent with the present invention is disclosed in WO 91/11457 (U.S. Patent No. 5,545,618) and consists essentially of GLP-1(7-34), GLP-1(7-35), GLP-1(7-36), or GLP-1(7-37), or the amide forms thereof, and pharmaceutically-acceptable salts thereof, having at least one modification selected from the group consisting of:

• (a) substitution of glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, arginine, or D-lysine for lysine at position 26 and/or position 34; or substitution of glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, lysine, or a D-arginine for arginine at position 36;
• (b) substitution of an oxidation-resistant amino acid for tryptophan at position 31;
• (c) substitution of at least one of: tyrosine for valine at position 16; lysine for serine at position 18; aspartic acid for glutamic acid at position 21; serine for glycine at position 22; arginine for glutamine at position 23; arginine for alanine at position 24; and glutamine for lysine at position 26; and
• (d) substitution of at least one of: glycine, serine, or cysteine for alanine at position 8; aspartic acid, glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, or phenylalanine for glutamic acid at position 9; serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, or phenylalanine for glycine at position 10; and glutamic acid for aspartic acid at position 15; and
• (e) substitution of glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, or phenylalanine, or the D-or N-acylated or alkylated form of histidine for histidine at position 7; wherein, in the substitutions (a), (b), (d), and (e), the substituted amino acids can optionally be in the D-form and the amino acids substituted at position 7 can optionally be in the N-acylated or N-alkylated form.

Because the enzyme, dipeptidyl-peptidase IV (DPP IV), may be responsible for the observed rapid in vivo inactivation of administered GLP-1, [see, e.g., Mentlein, R., et al., Eur. J. Biochem., 214:829-835 (1993)], the use of GLP-1 analogues and derivatives that are protected from the activity of DPP IV is required, and the use of Gly⁸-GLP-1(7-36)NH₂, Val⁸-GLP-1(7-37)OH, a-methyl-Ala⁸-GLP-1(7-36)NH₂, and Gly⁸-Gln²¹-GLP-1(7-37)OH, or pharmaceutically-acceptable salts thereof, is preferred. Also preferred is the use of a GLP-1 analogues selected from the group consisting of Val⁸-GLP-1(7-37)OH, Gly⁸-GLP-1(7-37)OH, and Asp⁸-GLP-1(7-37)OH.

The use of Val⁸-GLP-1(7-37)OH or a pharmaceutically-acceptable salt thereof, as claimed in US Patent Number 5,705,483, in the present invention is highly preferred.

Methods for preparing the GLP-1, GLP-1 analogues, or GLP-1 derivatives useful in the present invention are well-known in the art and are easily within the grasp of ordinarily skilled protein chemists or biochemists. The amino acid portion of the active compound used in the present invention, or a precursor thereto, can be made either by solid-phase synthetic chemistry, purification of GLP-1 molecules from natural sources, or recombinant DNA technology. Routine synthetic organic techniques enable the alkylation and acylation of the GLP-1 derivatives.

The term "GLP-1 related compound" refers to any compound falling within the GLP-1, GLP-1 analogue, or GLP-1 derivative definition.

The term "preservative" refers to a compound added to a pharmaceutical formulation to act as an anti-microbial agent. A parenteral formulation must meet guidelines for preservative effectiveness to be a commercially viable multi-use product. Among preservatives known in the art as being effective and acceptable in parenteral formulations are benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. See, e.g., Wallhauser, K., Develop. Biol. Standard, 24: 9-28 (Basel, S. Krager, 1974).

The term "buffer" or "pharmaceutically acceptable buffer" refers to a compound that is known to be safe for use in protein formulations and that has the effect of controlling the pH of the formulation at the pH desired for the formulation. Pharmaceutically acceptable buffers for controlling pH at a moderately acid pH to a moderately basic pH include, for example, such compounds as phosphate, acetate, citrate, TRIS, arginine, or histidine.

The term "isotonicity agent" refers to a compound that is tolerated physiologically and imparts a suitable tonicity to a formulation to prevent the net flow of water across the cell membrane. Compounds, such as glycerin, are commonly used for such purposes at known concentrations. Other acceptable isotonicity agents include salts and sugars, e.g., NaCl, dextrose, mannitol, and lactose. Glycerol at a concentration of 12 to 25 mg/mL is preferred as an isotonicity agent.

GLP-1 related compounds described above are administered by inhalation in a dose effective manner to introduce circulating therapeutic levels which results in reducing abnormally high blood glucose levels. Therapeutic serum levels of GLP-1 related compounds are in the range of 0.1 to 10.0 ng/ml, preferably 0.3 to 5.0 ng/ml and most preferably 0.5 to 3.0 ng/ml. Such administration can be effective for treating disorders such as diabetes, hyperglycemia, or insulin resistance. Achieving therapeutically effective doses of GLP-1 related compounds requires administering an inhalation dose of 0.5 µg/kg to 100 µg/kg of the GLP-1molecule, preferably 1.0 µg/kg to 50 µg/kg, more preferably 2.0 µg/kg to 25 µg/kg, and most preferably 2.5 µg/kg to 15 µg/kg. A therapeutically effective amount can be determined by a knowledgeable practitioner, who will take into account factors including native GLP-1 blood levels, blood glucose levels, the physical condition of the patient, the patient's pulmonary status, and the like.

According to the invention, GLP-1 analogues and derivatives are delivered by inhalation to achieve rapid absorption in the lungs. Administration by inhalation can result in pharmacokinetics comparable to subcutaneous administration of these substances. Inhalation of GLP-1 analogues and derivatives leads to a rise in the level of circulating insulin followed by a rapid fall in blood glucose levels. Different inhalation devices typically provide similar pharmacokinetics when similar particle sizes and similar levels of lung deposition are compared.

According to the invention, GLP-1 analogues and derivatives can be delivered by any of a variety of inhalation devices known in the art for administration of a therapeutic agent by inhalation. These devices include metered dose inhalers, nebulizers, dry powder inhalers, sprayers, and the like. Preferably, GLP-1 analogues and derivatives are delivered by a dry powder inhaler or a sprayer. There are a several desirable features of an inhalation device for administering GLP-1 analogues and derivatives. For example, delivery by the inhalation device is advantageously reliable, reproducible, and accurate. The inhalation device should deliver small particles, e.g. less than 10 µm mass median aerodynamic diameter (MMAD), preferably 1-5 µm MMAD, for good respirability. Some specific examples of commercially available inhalation devices, or those in late stage development, suitable for the practice of this invention are Turbohaler^™ (Astra), Rotahaler^® (Glaxo), Diskus^® (Glaxo), Spiros^™ inhaler (Dura), devices being developed by Inhale Therapeutics, AERx^™ (Aradigm), the Ultravent^® nebulizer (Mallinckrodt), the Acorn II^® nebulizer (Marquest Medical Products), the Ventolin^® metered dose inhaler (Glaxo), the Spinhaler^® powder inhaler (Fisons), or the like.

An inhalation device suitable for pulmonary administration and capable of depositing the GLP-1 molecule in the lungs of the patient when actuated can administer 40 µg to 4,000 µg of a GLP-1 molecule, preferably, 80 µg to 2,000 µg, more preferably, 160 µg to 1,000 µg, even more preferably, 320 µg to 500 µg.

As those skilled in the art will recognize, the formulation of GLP-1 analogues and derivatives, the quantity of the formulation delivered, and the duration of administration of a single dose depend on the type of inhalation device employed. For some aerosol delivery systems, such as nebulizers, the frequency of administration and length of time for which the system is activated will depend mainly on the concentration of the GLP-1 molecule in the aerosol. For example, shorter periods of administration can be used at higher concentrations of GLP-1 analogues and derivatives in the nebulizer solution. Devices such as metered dose inhalers can produce higher aerosol concentrations, and can be operated for shorter periods to deliver the desired amount of GLP-1 analogues and derivatives. Devices such as powder inhalers deliver active agent until a given charge of agent is expelled from the device. In this type of inhaler, the amount of GLP-1 analogues and derivatives in a given quantity of the powder determines the dose delivered in a single administration.

The particle size of the GLP-1 analogues and derivatives in the formulation delivered by the inhalation device is critical with respect to the ability of protein to deposit in the lungs, and preferably in the lower airways or alveoli. Preferably, the GLP-1 analogues and derivatives is formulated so that at least 10% of the peptide delivered is deposited in the lung, preferably 10% to 20%, or more. It is known that the maximum efficiency of pulmonary deposition for mouth breathing humans is obtained with particle sizes of 2 µm to 3 µm MMAD. When particle sizes are above 5 µm MMAD, pulmonary deposition decreases substantially. Particle sizes below 1 µm MMAD cause pulmonary deposition to decrease, and it becomes difficult to deliver particles with sufficient mass to be therapeutically effective. Thus, particles of GLP-1 analogues and derivatives delivered by inhalation have a particle size preferably less than 10 µm MMAD, more preferably in the range of 1 µm to 5 µm MMAD, and most preferably in the range of 2 µm to 3 µm MMAD. The formulation of GLP-1 analogues and derivatives is selected to yield the desired particle size in the chosen inhalation device.

Advantageously for administration as a dry powder, GLP-1 analogues and derivatives are prepared in a particulate form resulting in an emitted particle size less than 10 µm MMAD, preferably 1 to 5 µm MMAD, and most preferably 2 µm to 3 µm MMAD. The preferred particle size is effective for delivery to the alveoli of the patient's lung. Preferably, the dry powder is largely composed of particles produced so that a majority of the particles have a size in the desired range. Advantageously, at least 50% of the dry powder is made of particles having a diameter less than 10 µm MMAD. Such formulations can be achieved by spray drying, milling, or critical point condensation of a solution containing the particular GLP-1 molecule and other desired ingredients. Other methods also suitable for generating particles useful in the current invention are known in the art.

The particles are usually separated from a dry powder formulation in a container and then transported into the lung of a patient via a carrier air stream. Typically, in current dry powder inhalers, the force for breaking up the solid is provided solely by the patient's inhalation. One suitable dry powder inhaler is the Turbohaler^™ manufactured by Astra (Södertalje, Sweden). In another type of inhaler, air flow generated by the patient's inhalation activates an impeller motor which deagglomerates the GLP-1 molecule particles. The Dura Spiros^™ inhaler is such a device.

Formulations of GLP-1 analogues and derivatives for administration from a dry powder inhaler typically include a finely divided dry powder containing peptide, but the powder can also include a bulking agent, carrier, excipient, another additive, or the like. Additives can be included in a dry powder formulation of GLP-1 analogues and derivatives, for example, to dilute the powder as required for delivery from the particular powder inhaler, to facilitate processing of the formulation, to provide advantageous powder properties to the formulation, to facilitate dispersion of the powder from the inhalation device, to stabilize the formulation (e.g., antioxidants or buffers), to provide taste to the formulation, or the like. Advantageously, the additive does not adversely affect the patient's airways. The GLP-1 analogues and derivatives can be mixed with an additive at a molecular level or the solid formulation can include particles of the peptide mixed with or coated on particles of the additive. Typical additives include mono-, di-, and polysaccharides; sugar alcohols and other polyols, such as, for example, lactose, glucose, raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol, starch, or combinations thereof; surfactants, such as sorbitols, diphosphatidyl choline, or lecithin; or the like. Typically an additive, such as a bulking agent, is present in an amount effective for a purpose described above, often at 50% to 90% by weight of the formulation. Additional agents known in the art for formulating proteins can also be included in the formulation.

In another aspect of the invention a spray including GLP-1 analogues and derivatives can be produced by forcing a suspension or solution of the peptide through a nozzle under pressure. The nozzle size and configuration, the applied pressure, and the liquid feed rate can be chosen to achieve the desired output and particle size. An electro-spray can be produced, for example, by an electric field in connection with a capillary or nozzle feed. Advantageously, droplets of GLP-1 analogues and derivatives delivered by a sprayer have an inhaled droplet size less than 10 µm MMAD, preferably in the range of 1 µm to 5 µm MMAD, and most preferably 2 µm to 3 µm MMAD.

Formulations of GLP-1 analogues and derivatives suitable for use with a sprayer typically are 1 mg to 20 mg of the peptide per ml of solution. The formulation can include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and metal cations. The formulation can also include an excipient or agent to stabilize the peptide such as a buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulk proteins useful in formulating GLP-1 analogues and derivatives include albumin, protamine, or the like. Typical carbohydrates useful in formulating GLP-1 analogues and derivatives include sucrose, mannitol, lactose, trehalose, glucose, or the like. Formulations of GLP-1 analogues and derivatives can also include a surfactant, which can reduce or prevent surface-induced aggregation of the peptide caused by atomization of the solution in forming an aerosol. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between 0.001 and 4% by weight of the formulation. Other surfactants such as diphosphatidyl choline or lecithin can also be used. Especially preferred surfactants for purposes of this invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, or the like. Additional agents known in the art for formulating proteins can also be included in the formulation.

GLP-1 analogues and derivatives can be administered by a nebulizer, such as jet nebulizer or an ultrasonic nebulizer. Typically, in a jet nebulizer, a compressed air source is used to create a high-velocity air jet through an orifice. As the gas expands beyond the nozzle, a low-pressure region is created, which draws a solution of the peptide through a capillary tube connected to a liquid reservoir. The liquid stream from the capillary tube is sheared into unstable filaments and droplets as it exits the tube, creating the aerosol. A range of configurations, flow rates, and baffle types can be employed to achieve the desired performance characteristics from a given jet nebulizer. In an ultrasonic nebulizer, highfrequency electrical energy is used to create vibrational, mechanical energy, typically employing a piezoelectric transducer. This energy is transmitted to the peptide formulation either directly or through a coupling fluid, creating an aerosol. Advantageously, droplets of GLP-1 analogues and derivatives delivered by a nebulizer have a particle size less than 10 µm MMAD, preferably in the range of 1 µm to 5 µm MMAD, and most preferably 2 µm to 3 µm MMAD.

Formulations of GLP-1 analogues and derivatives suitable for use with a nebulizer, either jet or ultrasonic, typically include an aqueous solution of the peptide at a concentration of 1 mg to 20 mg per ml of solution. The formulation can include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and a divalent metal cation. The formulation can also include an excipient or agent to stabilize the peptide, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulk proteins useful in formulating GLP-1 analogues and derivatives include albumin, protamine, or the like. Typical carbohydrates useful in formulating GLP-1 related proteins include sucrose, mannitol, lactose, trehalose, glucose, or the like. Formulations of the GLP-1 analogues and derivatives can also include a surfactant, which can reduce or prevent surface-induced aggregation of the peptide caused by atomization of the solution in forming an aerosol. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbital fatty acid esters. Amounts will generally range between 0.001 and 4% by weight of the formulation. Other surfactants such as phosphatidyl choline or lethicin can also be used. Especially preferred surfactants for purposes of this invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, or the like. Additional agents known in the art for formulation of a protein such as GLP-1 related molecules can also be included in the formulation.

Another aspect of the invention involves a metered dose inhaler (MDI). In this embodiment, a propellant, GLP-1 analogues and derivatives, and any excipients or other additives are contained in a canister as a mixture including a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol, preferably containing inhaled particles in the size range of less than 10 µm MMAD, preferably 1 µm to 5 µm MMAD, and most preferably 2 µm to 3 µm MMAD. The desired aerosol particle size can be obtained by employing a formulation of GLP-1 analogues and derivatives produced by various methods known to those of skill in the art, including jet-milling, spray drying, critical point condensation, or the like. Preferred metered dose inhalers include those manufactured by 3M or Glaxo and employing a hydrofluorocarbon propellant.

Formulations of GLP-1 analogues and derivatives for use with a metered-dose inhaler device will generally include a finely divided powder containing peptide as a suspension in a non aqueous medium, for example, suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a (hydrofluroalkane-134a), HFA-227 (hydrofluroalkane-227), or the like. Preferably the propellant is a hydrofluorocarbon. The surfactant can be chosen to stabilize the GLP-1 molecule as a suspension in the propellant, to protect the active agent against chemical degradation, and the like. Suitable surfactants include sorbitan trioleate, soya lecithin, oleic acid, or the like. In some cases solution aerosols are preferred using solvents such as ethanol. Other surfactants such as diphosphatidyl choline or lethicin can also be used. Additional agents known in the art for formulating proteins can also be included in the formulation.

The present invention also relates to a pharmaceutical composition or formulation including GLP-1 analogues and derivatives and suitable for administration by inhalation. According to the invention, GLP-1 analogues and derivatives can be used for manufacturing a formulation or medicament suitable for administration by inhalation. The invention also relates to methods for manufacturing formulations including GLP-1 related molecules in a form that is suitable for administration by inhalation. For example, a dry powder formulation can be manufactured in several ways, using conventional techniques. Particles in the size range appropriate for maximal deposition in the lower respiratory tract can be made by micronizing, milling, spray drying, or the like. And a liquid formulation can be manufactured by dissolving the peptide in a suitable solvent, such as water, at an appropriate pH, including buffers or other excipients.

The present invention may be better understood with reference to the following examples. These examples are intended to be representative of specific embodiments of the invention.

The GLP-1 analogue, Val⁸-GLP-1(7-37)OH (SEQ ID NO:4) was prepared in E coli using conventional recombinant DNA techniques and purified to homogeneity.

One group of 6 female beagle dogs was exposed to inhaled Val⁸-GLP-1 for 15 minutes at an average aerosol concentration of 77.2 µg/L generated from a solution of Val⁸-GLP-1 in sterile water. These animals were dosed with 100 µg/kg via subcutaneous administration approximately 1-week after inhalation exposures. Tidal volume, breathing rate, and minute volume were monitored prior to and throughout the exposure period. Blood was collected for analysis of plasma levels of Val⁸-GLP-1 at various time points following inhalation and subcutaneous administration. Bronchoalveolar lavage (BAL) fluid was collected approximately 4 hours postexposure and analyzed for LDH, total protein, cell counts, and white cell differentials.

The delivery of Val⁸-GLP-1 was well tolerated with an inhaled dose of 1198 µg/kg and an estimated deposited lung dose of 240 µg/kg. Subcutaneous administration of 100 µg/kg was also well tolerated by all animals. Inhalation and subcutaneous administration of Val⁸-GLP-1 was delivered using formulated and formulated material.

There were no treatment-related clinical observations; body weights were not adversely affected by Val⁸-GLP-1. Only minor lung effects were observed. Increases in both tidal volume and minute volume were observed during the 15 minute inhalation exposures but the data were highly variable. No significant changes were observed for LDH, red blood cell counts, white blood cell counts, neutrophils, lymphocytes, eosinophils, epithelial cells, macrophages, basophils, or monocytes. There was a mild increase in total protein following aerosol delivery of Val⁸-GLP-1.

Results from this study demonstrated that there was good bioavailability of Val⁸-GLP-1 (40%, based on AUC) delivered to the lungs of beagle dogs by inhalation relative to subcutaneous administration. Val⁸-GLP-1 was well tolerated for up to 15 minutes with minimal effects on the lungs at an average inhaled dose of 1198 µg/kg, which was a no-observed-adverse-effects level (NOAEL) in this study.

Solutions of Val⁸-GLP-1 were prepared on the days of dosing at concentrations of 0.5 mg/ml or 8 mg/ml in sterile water for subcutaneous administration and pulmonary administration, respectively. An additional solution of Val⁸-GLP-1 (8 mg/ml) was prepared at the end of live phase in order to determine the particle size distribution of aerosolized Val⁸-GLP-1. The solutions were filtered through a low protein binding 0.22 micron canister filter. The pH of the solution was adjusted with sodium hydroxide solution to 7.47.

Six females Beagle dogs (Marshall Farms, North Rose, NY) were used in this study. Each animal was uniquely identified by a five-digit animal number and a seven-digit tattoo (located on inner ear) number recorded on their cage card. All of the animals were acclimated to the restraint slings prior to beginning the study. The weight range of the animals at the start of the study was 8.4 to 11.1 kg. The age of the animals at the start of the study was 33 to 37 weeks.

Animals were pair housed in stainless steel cages except on the days of exposure. Each animal was individually housed on the day of exposure in order to monitor their feeding regimen. Rooms were thermostatically set to maintain a temperature of 70°F and maintain the actual temperature within ±8°F from that set point. The environmental control system is designed to maintain a relative humidity of 20% and a maximum of 80%. Light was on a 12-hour cycle, with lights on between 0600 and 1800 hours. Subsequently, lights were off between 1800 and 0600 hours except when blood samples were collected. Animals were fed once daily with Hill's Science Diet. Animals were fasted for approximately 12-hours prior to exposure. Tap water was provided ad libitum except during exposures.

All 6 dogs were exposed for 15 minutes to aerosolized Val⁸-GLP-1. The targeted deposited lung dose for Val⁸-GLP-1 exposures was 200 µg/kg of body weight. Approximately 7 days after pulmonary administration of Val⁸-GLP-1, all dogs were dosed with 100 µg/kg Val⁸-GLP-1 of body weight via subcutaneous administration.

The dogs were tested while standing in restraint slings. Two layers of 0.762 millimeters (0.03 inch) latex sheets were placed around the animals' necks to form a nonrestrictive airtight seal. A custom built, 11-L head-dome, similar to that described by Allen et al. (J Appl Toxicol 1995; 15:13-17) was placed over the dogs' heads and secured to the sling. Airflow was exhaust driven via a transvector located on the exhaust side of the dome. Because the helmet was airtight and the neck was sealed, this constituted a head-only exposure system. The total flow rate through the dome was approximately 7.5 L/min.

The aerosols were generated using a Respirgard II nebulizer with an input of approximately 6.5 L/min. The output from the generator flowed directly into the head-dome.

All sampling for total gravimetric concentrations was performed with in-line filter holders containing type A/E glass fiber filters (Gelman Instruments Co., Ann Arbor, MI). Filters sampling from the chamber was carried out at a nominal sampling rate of 1 L/min, calibrated with a portable mass airflow calibrator (Model 830, Sierra Instruments, Carmel Valley, CA). Sampling duration was 15 minutes. Particle size analysis was performed with a Sierra Model 218K Ambient Cascade Impactor (Anderson Samplers, Inc, Atlanta, GA). Cascade sampling from the chamber was carried out at a nominal sampling rate of 3 L/min, calibrated with a portable mass airflow calibrator (Model 830, Sierra Instruments, Carmel Valley, CA). Sampling duration was 31 minutes. Filters were allowed to dry for approximately 30-minutes before being re-weighed.

The dose of Val⁸-GLP-1 inhaled during a 15-minute exposure was estimated as follows: the mean minute volume (mL) during the 15 minute exposure was multiplied by the exposure duration to yield the total air breathed (L) during the inhalation exposure. This value was multiplied by the aerosol concentration (µg/L) to determine total dose (mg). The inhaled dose (µg/kg) was calculated by dividing the total dose (µg) by the animal's body weight (kg).

The dose of Val⁸-GLP-1 deposited in the lungs was estimated as follows: inhaled dose (µg/kg) was multiplied by 20 percent to yield estimated deposited lung dose (µg/kg). Aerosols with a MMEAD ranging from 1-2 µm MMAD have been shown to deposit in the lung with approximately a 20 percent efficiency (Schlesinger RB. 1985. Comparative deposition of inhaled aerosols in experimentalanimals and humans: a review. J Toxicol Environ Health 15:197-214).

All animals were weighed on Days -5, 0, and 7. Breathing patterns (tidal volume, breathing frequency, and minute volume) were monitored using a size '0' pneumotachograph connected to a port on the head-dome. The signals were collected on a personal computer using the Buxco XA Data Acquisition System (Buxco Electronics, Inc, Sharon, CT). At least 15 minutes of preexposure data were collected before the exposures began, followed by data collection throughout the 15-minute exposure periods. All data were analyzed as 5-minute averages.

Bronchoalveolar lavage (BAL) was performed approximately 4 hours after each dosing regimen. The animals were anesthetized with an intravenous injection of 2% Brevital prior to the BAL procedure. Bronchoalveolar lavage was performed using a pediatric fiberoptic bronchoscope (Olympus, model BF, Type 3C10, Lake Success, NY). The bronchoscope tip was wedged in a 5^th to 7^th generation airway of a lower lobe. The BALs were alternated between the right and left lung lobes. Two 10-mL aliquots were instilled and gently suctioned out. Aliquots of the recovered lavage fluid were used to determine total white cell counts, total red cell counts, white cell differentials, total protein, and lactate dehydrogenase.

A complete blood count was done on the unconcentrated BAL fluid using a Technicon H1 System (Technicon Instruments Corporation). Cell differentials for the BAL were done by microscopic evaluation of 200 Wright stained cells.

For analysis of plasma pharmacokinetics of Val⁸-GLP-1, approximately 2 to 3 mL of blood were collected in EDTA vacutainer tubes from the cephalic or jugular vein prior to exposure and at 0.25, 0.5, 1, 2, 3, 4, 6, 8, and 12 hours postexposure. To obtain the plasma, each tube was centrifuged at approximately 3000 rpm for 10 minutes at 10°C. The plasma samples were stored at -70°C until sent for assay.

The average exposure concentration for each dog exposed to Val⁸-GLP-1 ranged from 64.0 to 101.3 µg/L. The mean (+ SD) concentration for all animals was 77.2 ± 16.9 µg/L. Particle size was measured as a mass median equivalent aerodynamic diameter (MMEAD) of 0.91 µm with a geometric standard deviation (GSD) of 2.37.

The average dose of Val⁸-GLP-1 deposited in lungs of 6 dogs treated with Val⁸-GLP-1 was calculated as 240 ± 42 µg/kg (mean + SD). The mean inhaled dose (that which entered the respiratory tract neglecting deposition) was 1198 ± 208 µg/kg (mean ± SD). Individual animal data are shown in the table below. Animal Number Aerosol Conc. (µg/L) Average Minute Volume (ml) Inhaled Lung Dose (µg/kg) Deposited Lung Dose (µg/kg) 27682 0.07717* 8406 1060 212 27684 0.07717* 9563 1030 206 27685 0.10133 7260 1350 270 27686 0.06400 12733 1360 272 27687 0.06733 9214 950 190 27689 0.07600 11890 1400 288 *Aerosol concentration was not determined for animals 27682 and 27684, therefore the mean aerosol concentration was used to calculate an estimated inhaled lung dose (µg/kg).

No significant changes in body weight were observed during the treatment phase. The initial (Day -5) and final (Day 7) body weights were 9.5 ± 0.9 (mean ± SD; n=6) and 9.7 ± 0.9 kg, respectively. No marked changes in breathing frequency was observed. Slight increases in tidal volume and minute volume were measured but the data was highly variable potentially due to the short acclimation period prior to study initiation. Individual animal data are shown in the table below. Animal Tidal Volume (mL) Breathing Frequency (bpm) Minute Volume (mL/min) Number 5* 10* 15* 5* 10* 15* 5* 10* 15* 27682 119.0 152.1 105.1 74 86.0 95.7 7265 9360 8592 27684 104.6 99.4 100.8 119.2 118.2 108.7 10300 9117 9272 27685 165.9 178.5 209.6 58.1 49.4 42.5 7938 7465 6377 27686 325.1 586.2 374.4 56.3 33.8 59.4 12200 13300 12700 27687 245.9 221.6 183.1 39.5 45.5 50.5 9288 9573 8781 27689 184.5 367.6 454.0 39.8 46.8 33.3 7271 13400 15000 Mean 190.8 267.6 237.8 64.6 63.3 65.0 9044 10369 10120 Stdev 82.8 180.7 145.3 29.8 32.2 30.3 1956 2426.1 3141 Mean 239.0 216.8 207.6 45.9 51.4 64.4** 8448 8744.8 8223** (baseline) ** Stdev 161.4 137.2 209.5 12 10.2 32.3 2614 2614 1566 (baseline) *minutes during exposure (values represent average over 5 minutes)
**mean calculated from a n=5 (instead of n=6) because no value was recorded for animal
#27689 at 15 minutes preexposure Delivery Route C_max (ng/mL) T_max (h) AUC_0-t(1) (ng*h/mL) T1/2(α) (hours) Subcutaneous 10.53 ± 1.06 0.71 ± 0.14 36.37 ± 2.18 1.26 ± 0.11 Inhalation 8.66 ± 0.90 1.54 ± 0.59 35.21 ± 5.91 1.19 ± 0.11 *All values are reported as mean ± SEM (standard error of the mean).
₍₁₎ AUC_0-t'=area under the plasma concentration curve from time 0 to t, where t=12 hours postdose

Plasma concentrations of immunoreactive Val⁸-GLP-1 (Table 3) were measured by a competitive radioimmunoassay (RIA). The absorption of Val⁸-GLP-1 via both delivery routes appeared to be rapid, reaching substantial plasma concentrations at 15 minutes postdose. The plasma time profiles were similar for the subcutaneous injections and inhalation. The average Tmax value for inhalation was greater than that for subcutaneous injection. Also, the elevated plasma Val⁸-GLP-1 concentration (close to Cmax) achieved by inhalation appeared to remain near that level for a longer period of time than following subcutaneous injection.

Based on the average AUC values, the bioavailability of inhaled Val⁸-GLP-1 (averaged inhaled dose of 1198 µg/kg) relative to subcutaneous injection (100 µg/kg) was approximately 7.7%. On the basis of deposited lung dose, estimated as 240 µg/kg, the bioavailability relative to subcutaneous injection was 40%.